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| A physical map of human Alu repeats cleavage by restriction endonucleases |
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| | Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico.
Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier.
Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.
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| Substrate specificity of new methyl-directed DNA endonuclease GlaI |
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| | Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI. In a recent work we have studied the dependence of GlaI activity on the quantity and location of 5-methylcytosines in the enzyme recognition sequence 5'-GCGC-3'/3'-CGCG-5'. A significant DNA cleavage has been observed for oligonucleotide duplexes, which include either three or four 5-methylcytosines. In this work we have studied dependence of the GlaI activity on quantity and location of methylated cytosines, as well as on composition of the recognition sequence.The list of good substrates for GlaI includes a fully methylated site 5'-CGCG-3'/3'-GCGC-5', sites with three cytosines of a general structure 5'-PuMGM-3'/3'-PyGMG-5', and one recognition sequence with two methylated cytosines 5'-AMGT-3'/3'-TGMA-5', where M is 5-methylcytosine. GlaI intermediate substrates include sites with three methylated cytosines of a general structure 5'-GMPuM-3'/3'-MGPyG-5', as well as a site with two methylcytosines 5'-GMGT-3'/3'-CGMA-5'. The site 5'-GMGC-3'/3'-CGMG-5' may be considered a low activity substrate. | |
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| Comparative analysis of human chromosomal DNA digestion with restriction endonucleases in vitro and in silico. |
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| | Theoretical analysis of human chromosomal DNA cleavage at 15 nucleotide sequences, which are the recongnition sites of various restriction endonucleases, has been carried out. Distribution diagrams of calculated DNA fragments have been constructed based on earlier proposed method of mammalian genomes digestion in silico. A similar study of human Alu- and LINE1-repeats nucleotide sequences, which are present in informational databases, has been performed and corresponding diagrams of DNA fragments distribution have been plotted. Distribution diagrams of chromosomal DNA digestion, which results in formation of low molecular weight DNA fragments, correspond to those for Alu-repeats; whereas the digestion, which results in formation of large molecular weight DNA fragments - are similar to those for LINE-repeats. All theoretical data have been compared to experimental patterns of human genomic DNA cleavages with respective restriction endonucleases and a good correspondence for the most of DNA diagrams has been observed. | |
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| A novel site-specific endonuclease GluI recognizes methylated DNA sequence 5-G(5mC)^NG(5mC)-3/3-(5mC)GN^(5mC)G. |
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| | A novel site-specific endonuclease GluI from the bacterial strain GL24 has been isolated and characterized. The enzyme recognizes methylated DNA sequence 5-G(5mC)^NG(5mC)-3/3-(5mC)GN^(5mC)G-5 and cleaves it as it is shown by arrow. Due to its ability to cleave only modified DNA GluI may be useful for genetic engineering experiments as well as for determination of DNA methylation status in eucaryotes. | |
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| Site-specific endonuclease BlsI recognizes DNA sequence 5-G(5mC)N^GC-3 and cleaves it producing 3 sticky ends |
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| | A bacterial strain Bacillus simplex 23, a producer of a site-specific endonuclease BlsI has been discovered. BlsI recognizes the methylated DNA sequence 5-G(5mC)N↓GC-3, like the earlier described site-specific endonuclease BisI (recognition site 5-G(5mC)↓NGC-3), but differs in positions of DNA cleavage producing 3-protruding ends. Due to its ability to cleave only methylated DNA, enzyme BlsI can find an application in gene engineering works as well as in determining the methylation status of eucaryotic DNA. | |
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| New Restriction Endonuclease AluBI from Arthrobacter luteus B - AluI Isoshizomer, nonsensitive to Presence of 5-methylcytosine in the Recognition Sequence AGCT |
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| | A new restriction endonuclease AluBI has been discovered and characterized. AluBI is an isoshizomer of well known restriction endonuclease AluI, which recognizes DNA sequence AGCT. Unlike AluI, enzyme AluBI is able to cleave DNA when recognition sequence contains 5-methylcytosines. AluBI also cleaves recognition sequence with two methylated cytosines, one modified at N4 and other at C5 positions. However, new enzyme doesnt cleave DNA with two N4-methylcytosines in the recognition site. | |
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| A novel restriction endonuclease GlaI recognizes methylated sequence 5-G(m5C)^GC-3 |
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| | A novel restriction endonuclease GlaI from the bacterial strain GL29 has been isolated and characterized. The enzyme recognizes methylated DNA sequence 5-G(5mC)↓GC-3 and cleaves it as indicated by arrow. Due to its ability to cleave only modified DNA GlaI may find a practical application in genetic engineering experiments as well as in determination of DNA methylation status. | |
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| Comparative restriction enzymes analysis of rat chromosomal DNA in vitro and in silico |
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| | Theoretical diagrams of rat chromosomal DNA cleavage at more than 25 different nucleotide sequences have been plotted based on earlier proposed method of restriction enzymes analysis of mammalian genomes in silico. A map of the rat LINE1 repeats digestions at the same nucleotide sequences has been calculated and a correspondence between the diagrams data and LINE1 repeat cleavage positions has been shown. Restriction enzymes analysis in vitro has been performed in order to obtain patterns of rat chromosomal DNA cleavage by endonucleases with respective recognition sequences. A perfect coincidence has been shown between theoretical DNA cleavage diagrams and experimental patterns of DNA hydrolysis with restriction endonucleases. | |
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| Mammalian chromosomal DNA digestion with restriction endonucleases in silico |
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| | A theoretical method to produce digestion patterns of mammalian chromosomal DNA cleavage by restriction endonucleases was proposed. Based on recently published data on primary structures of genomes, a computer analysis was performed and diagrams of chromosomal DNA fragments distribution were plotted for DNA cleavages at 5-GGCC-3', 5'-CCGG-3', 5'-GATC-3' and 5'-CC(A/T)GG-3' sequences. Experiments on chromosomal DNA digestion with HaeIII, MspI, Kzo9I and Bst2UI restriction endonucleases, which recognize these sites, were carried out. A correspondence between the computed diagrams and experimentally observed patterns of restriction enzymes cleavage was shown. | |
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| New Eight Bases Cutter AbsI from Arthrobacter species Recognizes Palindromic DNA Sequence 5-CC^TCGAGG-3 |
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| | Bacterial strain Arthrobacter species7M06 producing site-specific endonuclease AbsI has been discovered. AbsI recognizes palindromic octanucleotide DNA sequence 5-CC^TCGAGG-3 and hydrolyzes it after second cytosine, producing 5- sticky ends, which are compatible with sticky ends after DNA cleavage by restriction endonucleases XhoI (5-C^TCGAG-3), PspXI (5-VC^TCGAGB-3) and SalI (5-G^TCGAC-3). Among all known rare-cutting site-specific endonucleases AbsI is the only enzyme which has no recognition sequences in standard substrates Lambda and T7 DNAs and Adenovirus type 2 DNA | |
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| Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5-GCGC-3. |
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| | The activity dependence of site-specific endonuclease GlaI that recognizes and hydrolyzes only methylated DNA sequence 5-GCGC-3 on the quantity and location of 5-methylcytosines in enzymes recognition sequence has been studied. A significant DNA cleavage has been observed for oligonucleotides duplexes containing four and three 5-methylcytosines or two internal modified bases. The cleavage efficiency is maximal for DNA duplex with four 5-methylcytosine and decreases when a number of methylated bases are lower. GlaI hydrolyzes recognition sequences with 5-methylcytosines but not with N4-methylcytosines. | |
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| An improved ARDRA method of microorganism identification and its application in identifying thermolabile alkaline phosphatase strains-producers |
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| | Based on the analysis of amplified ribosomal DNA fragmentation by restriction endonucleases, an improved method for microorganism identification (called ARDRA) has been suggested. A set of 6 restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI and RsaI) was used to get corresponding patterns of DNA cleavage. DNA samples were isolated from four strain-producers of thermolabile alkaline phosphatase, obtained from sea water. Carrying out of ARDRA, followed by a comparison with the calculated cleavage patterns of DNA from several referenced microorganisms, allowed us to conclude that the new strains-producers belong to the Alteromonas genus. | |
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| PspXI, a novel restriction endonuclease that recognizes the unusual DNA sequence 5-VC↓TCGAGB-3 |
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| | We have discovered a bacterial strain Pseudomonas species X11 that produces the unique restriction endonuclease PspXI. This enzyme recognizes the degenerate octanucleotide sequence 5-VCTCGAGB-3, where V stands for A, C or G and B stands for T, C or G.
The PspXI restriction endonuclease preparation with the specific activity of 10000 units/ml was isolated using four chromatographic steps. PspXI cuts its recognition sequence between C and T producing cohesive ends compatible with those of produced by XhoI and SalI restriction endonucleases.
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| Restriction endonuclease Bis I from Bacillus subtilis T30 recognizes methylated sequence 5-G(m5C)↓NGC-3 |
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| | Bacillus subtilis strain T30, producing a novel restriction endonuclease Bis I, has been isolated and characterized. The enzyme recognizes methylated DNA sequence 5-G(5mC)↓NGC-3 and cleaves it as it is shown by arrow. Due to cleavage of only modified DNAs Bis I may find a practical application in genetic engineering experiments as well as in determination of eukaryotic DNA methylation status. | |
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| Restriction Endonuclease AjnI from Acinetobacter johnsonii R2, an Isoschizomer of EcoRII, recognizes 5'- CCWGG-3' and cleaves dcm-methylated sites |
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| | A producer of restriction endonuclease AjnI has been isolated from natural resources and identified as bacterial species Acinetobacter johnsonii R2. The restriction enzyme purification and estimation of its activity is described. It has been shown that AjnI produces DNA fragments with 5'-CCWGG sticky ends similar to EcoRII, but cleavage is not blocked by dcm-methylation. A new restriction endonuclease AjnI may be widely used in genetic engineering. | |
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| A new site-specific DNA nickase N.Bst9I from Bacillus stearothermophilus 9 |
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| | Bacillus stearothermophilus 9 strain has been found during screening of
bacteria from natural sources which produces a new site-specific nickase
N.Bst9I. This enzyme recognizes and cleaves nucleotide sequence 5'-GAGTCNNNN^-3',
so it is an isoschizomer of N.BstSEI endonuclease found earlier. But enzymatic
properties of both enzymes are considerably different. N.Bst9I application in
genetic engineering and biotechnology is more preferable because its "star"
activity is much lower in comparison to N.BstSEI.
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| The Second Methyltransferase of the BstF5I RestrictionModification System Is Homologous to the C-Terminal Domains of FokI and StsI Methylases |
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| | The bstF5IM-2 gene for the second DNA methyltransferase of Bacillus stearothermophilus F5 (M.BstF5I-2) was cloned and sequenced. On the chromosome, the gene is located downstream of the gene for M.BstF5I-1 and is oriented similarly. The protein encoded has conserved regions specific for adenine methylases of subclass D12. M.BstF5I-2 is homologous to the C-terminal domains of M.FokI and M.StsI and recognizes the same sites. M.BstF5I-1 modifies the DNA upper strand and M.BstF5I-2 modifies the other strand in the recognition site. | |
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| BtrI, a novel restriction endonuclease, recognises the non-palindromic sequence 5'-CACGTC(-3/-3)-3' |
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| | The recognition sequence and cleavage positions of a new restriction endonuclease BtrI isolated from Bacillus stearothermophilus SE-U62 have been determined. BtrI belongs to a rare type IIQ of restriction endonucleases, which recognise non-palindromic nucleotide sequences and cleave DNA symmetrically within them.
Type II restriction endonucleases (ENases) include a group of 53 prototypes that recognise non-palindromic DNA sequences (ENases with recognition sequences that are interrupted by more than one base pair are not considered). Cleavage positions have been determined for 45 of these [1]. Mainly, such ENases cleave DNA outside their recognition sites and are designated type IIS [2]. There are only a few so-called type IIQ restriction enzyme prototypes that cut both DNA strands symmetrically within their non-palindromic recognition sequences [3].
Here we report a new member of this rare subgroup of type II ENases.
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| Multiplicity of Site-specific DNA Methyltransferases of the BstF5l Restriction-Modification System |
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| | A fragment located downstream of the genes for DNA methyltransferases of Bacillus stearothermophilus F5 (M.BstF5I-1 and M.BstF5I-2) was sequenced. The fragment contains a gene for another methylase. M.BstF5I-3. structurally and functionally similar to the N-terminal domain of M.FokI. Thus, in contrast to other restriction-modification systems, the BstF5I system includes three methylases, two being homologous to the individual M.FokI domains. | |
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| PsiI, a unique Restriction Endonuclease Recognizing the DNA Sequence 5'-TTA^TAA-3' |
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| | PsiI, a new restriction endonuclease produced by the bacterial strain Pseudomonas sp. SE-G49, has been isolated and characterized. The enzyme cleaves DNA in the middle of its palindromic recognition sequence 5-TTA^TAA-3'. Thus, PsiI belongs to a rare group of type II restriction endonucleases whose recognition sites consist of AT base pairs only. | |
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| AccBSI: A New Restriction Endonuclease from Acinetobacter calcoaceticus BS |
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| | The recognition site of a new restriction endonuclease from Acinetobacter calcoaceticus BS was determined. This is a nonpalindromic sequence
5'-GAGCGG-3'
3'-CTCGCC-5'
AccBSI restriction endonuclease cleaves DNA chains in the middle of the recognition sequence; therefore, ligation of its digestion fragments restores AccBSI recognition sites and generates palindromic sequences recognized by SacI and SacII restriction endonucleases. | |
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| N.BstSE, Site-Specific Nickase from Bacillus stearothermophilus SE-589 |
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| | A site-specific nickase recognizing and cleaving the DNA site
5'-G-A-G-T-C-N-N-N-N^N-N-3'
3'-C-T-C-A-G-N-N-N-N-N-N-5'
was isolated from Bacillus stearothermophilus SE-589 and named N.BstSE. Its properties indicate a possible relation with type II restriction endonucleases.
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| The resctriction endonucleases detection in colonies of microorganisms Streptomyces and Nocardia |
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| | A simple technique is proposed for detection of restriction endonucleases in bacteria Streptomyces and Nocardia. The analysis was performed directly in the cells cultivated on Petri dishes and collected from colonies by an inoculation loop. The cells were treated with lysozyme, EDTA and Triton X-100. The lysates were tested for restriction endonucleases. The technique enables todetect the enzymes Nco I, Not I, Nru I I, Sfr 3031, and Sfi I in the lysates of corresponding strains-producers. | |
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| A simple technique for detection of restriction endonucleases in bacterial colonies |
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| | A simple technique is proposed for detection of bacterial restriction endonucleases. Analysis is performed directly in the cells from colonies cultivated on Petri dishes. The cells are collected by a microbiological loop into microtubes and then are treated with lysozyme and Triton X-100. After centrifugation of bacterial lysat the supernatant is tested for a presence of site-specific endonuclease activity. The technique enables to analyze about 100 colonies for 3-4 hours. | |
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